CN105750311A - Comprehensive bioremediation method of lead and cadmium composite contaminated soil - Google Patents
Comprehensive bioremediation method of lead and cadmium composite contaminated soil Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
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Abstract
The invention discloses a comprehensive bioremediation method of lead and cadmium composite contaminated soil. For the lead and cadmium composite contaminated soil, the method mainly adopts hyper-accumulator remediation and is assisted by microbial remediation and animal remediation which can improve the phytoremediation effect, the bio-availability of lead and cadmium in the soil is enhanced, thus growth and absorption of the hyper-accumulators are promoted, and the comprehensive remediation efficiency of the contaminated soil is improved. In-situ bioremediation of the soil is realized smoothly, the effect is obvious, no secondary pollution is caused to the environment, and the method is the development direction in the remediation field of heavy metal contaminated soil in the future.
Description
Technical field
The present invention relates to the biological restoration of soil pollution, the comprehensive organism recovery technique of especially a kind of plumbous cadmium combined pollution soil.
Background technology
Soil is one of natural resources most basic, most important of depending on for existence and development of the mankind, is also important component part and the filter of human ecological environment, however the soil pollution of China the situation is tense.Along with the quickening of China's industrialization and urbanization process, the multiple pollutant containing heavy metal enter soil by all means, cause heavy metal pollution of soil day by day serious.According to the national irrigating region investigation that the Ministry of Agriculture of China carries out, at about 1,400,000 hm2Irrigating region in, suffer the land area of heavy metal pollution to account for the 64.8% of the irrigating region gross area, wherein slight pollution account for 46.7%, intermediate pollution account for 9.7%, severe contamination to account for 8.4%(Chen Zhi good, 2002).At present, the cultivated area of whole nation heavy metal pollution reaches 20,000,000 hm2, accounting for the 1/5 of total cultivated area, cause that the content of beary metal having 12,000,000 t grains every year exceeds standard, and cause grain drop in production 10,000,000 t, both direct economic losses reach more than 200 hundred million yuan (paddy Zhao, 2008).Additionally, China also has the pollution (Wang Zhilou, 2010) by heavy metal of the bare places such as a large amount of industrial and mining area soil and urban land.
In The Soils of Hebei heavy metal pollution situation allows of no optimist, plumbous, cadmium pollution outstanding problem.According to Hebei province's environmental aspect publication in 2004, the whole province's sewage irrigation area is 53277 hectares, focus on 7 rivers such as the Hutuohe River of Ziyahe River water system, river, oxtail river, vast sea ditch, Fu Yanghe, azure water canal, row river, north, relate generally to Shijiazhuang, Xingtai, Handan, Hengshui, Cangzhou 5 city.Wang Yuanzhong etc. 2005 are to county of Hebei province 15 20.53 ten thousand hm2Semen Tritici aestivi, farmland, main maize area 1535 pedotheques in the pollution situation of heavy metal the survey showed that: all there is pollution accumulation phenomenon in various degree in main maize area, Hebei province in 8 heavy metal species of main maize area, Hebei province;The whole province's gross contamination area has accounted for the 12.0% of the monitoring gross area.The research of the river irrigating region to Hebei province in 2004 such as Meng Fanqiao and river, mansion irrigating region shows, through the dirty filling of 20 years, in the soil in Fu He, He Liang great blowdown district, Cd exceeded the secondary standard of China's " standard of soil environment quality ", has obvious pollution.Shijiazhuang City irrigating region upper, middle and lower are swum the farmland topsoil contents of heavy metal elements in 3 sections, pollution situation, form etc. in 2009 and have been carried out investigation and research by Luan Wenlou etc..Found that: in irrigating region upper, middle and lower, trip all has enrichment in various degree to the heavy metal (except As) in the topsoil of study area, wherein Hg, Cd content is higher, in the topsoil of downstream heavy metal average content be above, middle reaches, downstream Cd average content is 8.51 times of In The Soils of Hebei background value, for 8.25 times of whole nation soil background.
The repairing and treating of heavy-metal contaminated soil is current international study hotspot, and the major issue solved currently is badly in need of in Ye Shi China.Soil once contaminated by heavy metals, just have the feature such as seriousness of irreversibility, disguise and consequence, not only may result in the decline of crop yield and quality, more can enter human body by food chain and bring serious harm (Nabulo, G., 2006 to human health；Ma Zhihong, 2007).The heavy metal of contaminated soil environment is primarily referred to as the significant cadmium of bio-toxicity, hydrargyrum, lead and metalloid arsenic.IARC (IARC) classifies as first kind human carcinogen (Li Zhaohui, 2010) cadmium.The plumbous nervus centralis mainly by murder by poisoning human body, causes the damage of the multiple organ such as system, liver spleen to health, especially the youngsters and children of future generation of motherland is endangered more very (Luo Ya, 2012；Wu Qianqian, 2014).Therefore, the repairing and treating of heavy-metal contaminated soil is the study hotspot outside Now Domestic.Especially after China joined WTO, the heavy metals exceeding standard problem of agricultural product China's agricultural will be produced large impact more by the trade barrier caused, the improvement of heavy metal pollution of soil is the major issue that China is currently badly in need of solving.
Domestic and international heavy metal repairing research field development process with initial reason, change restorative procedure and develop into biological restoration, and to comprehensive repair trend development.Domestic and international expert has carried out big quantity research in succession, wherein in early days based on traditional chemically and physically administering method (Renella.G, 2002), Europe and North America many developed countries 20th century the mid-80 carried out bioremediation technology preliminary study work (Nabulo, G.,, and complete the process engineering of some practicalities 2006).Biological restoration (bioremediation) technology is increasingly becoming the emerging environmental organism hot spot technology that countries in the world are paid close attention in recent years, the concept of broad sense refers to and utilizes biological vital metabolic activity reduce the concentration of poisonous and harmful substances in environment or make it completely innoxious, so that polluted environment can partially or even wholly return to the process (Zhou Qixing of initial condition, Wei Shu and, 2007).The biological restoration of narrow sense, including microorganism remediation and phytoremediation, it be primarily referred to as utilize biotechnology the hard-degraded substance such as macromole organic pollution, the heavy metal etc. that enter in soil environment are administered (Yan Xiaoming, what principal column., 2002).
In sum, the reparation of heavy-metal contaminated soil is a system engineering, and in natural ecosystems, mainly there is (Han Guiqi, Xu Weihong, 2010) with combined pollution in heavy metal pollution, single recovery technique is extremely difficult to Expected Results (Cui Dejie, Zhang Yulong, 2004).
Summary of the invention
The technical problem to be solved in the present invention is all deficiencies overcoming prior art, it is provided that a kind of efficient, economic plumbous cadmium combined pollution soil comprehensive organism repairing method.
For solving above-mentioned technical problem, the technical solution used in the present invention is as follows.
A kind of plumbous cadmium combined pollution soil comprehensive organism repairing method, soil for plumbous cadmium combined pollution, based on over-accumulated plant reparation, it is aided with the microorganism remediation that can improve effect of plant restoration and animal is repaired, strengthen Lead In Soil, the biological effectiveness of cadmium and thus promote growth and the absorption of plant, the overall efficiency of raising contaminated soil remediation.
As a preferred technical solution of the present invention, the method adopts phytoremediation to add animal and repairs the combined remediation method adding microorganism remediation.
As a preferred technical solution of the present invention, described phytoremediation adopts arabian cron, and described animal is repaired and adopts Lumbricus, and described microorganism remediation adopts the composite bacteria agent capable containing sulfur bacteria, nitrifier, Ammonifying bacteria and nitrogen-fixing bacteria；Described plant, animal, microorganism, combine d bioremediation operating procedure be: arabian cron sow before, keep soil moisture 50%-70% by shallow for the composite bacteria agent capable 3-5cm of turning over mixing be manured into soil；Then, after arabian cron Seedling all sprouts, temperature throws in Lumbricus at 18-25 DEG C and soil moisture when 50%-70%.
As a preferred technical solution of the present invention, first measure the pollution level of soil, and soil contamination grade delimited as slight pollution, intermediate pollution or serious pollution；If soil belongs to slight pollution, then adopt phytoremediation mode or employing phytoremediation to add animal and repair the combine d bioremediation mode adding microorganism remediation；If soil belongs to intermediate pollution, then adopt phytoremediation mode or employing phytoremediation to add animal and repair the combine d bioremediation mode adding microorganism remediation；If soil belongs to serious pollution, then adopt phytoremediation to add animal and repair the combine d bioremediation mode adding microorganism remediation.
As a preferred technical solution of the present invention, the demarcation method of described soil contamination grade is as follows: the lead content in soil is measured, if lead content is less than 300mg/kg, then delimit as slight pollution, if lead content is more than 300mg/kg and less than 600mg/kg, then delimiting is intermediate pollution, if lead content is more than 600mg/kg, then delimits as serious pollution.
As a preferred technical solution of the present invention, the cadmium content in soil is measured, if cadmium content is less than 1.5mg/kg, then delimit as slight pollution；If cadmium content is more than 1.5mg/kg and less than 6mg/kg, then delimit as intermediate pollution；If cadmium content is more than 6mg/kg, then delimit as serious pollution.
As a preferred technical solution of the present invention, described phytoremediation adopts arabian cron, and described animal is repaired and adopts Lumbricus, and described microorganism remediation adopts the composite bacteria agent capable containing sulfur bacteria, nitrifier, Ammonifying bacteria and nitrogen-fixing bacteria；Described plant, animal, microorganism, combine d bioremediation operating procedure be: arabian cron sow before, keep soil moisture 50%-70% by shallow for the composite bacteria agent capable 3-5cm of turning over mixing be manured into soil；Then, after arabian cron Seedling all sprouts, temperature throws in Lumbricus at 18-25 DEG C and soil moisture when 50%-70%.
As a preferred technical solution of the present invention, described phytoremediation adopts common over-accumulated plant kind or adopts transgenic Sudan. grass Varieties.
As a preferred technical solution of the present invention, the preparation process of described transgenic Sudan. grass Varieties includes:
A, receptor preparation: choose full neat arabian cron seed, clean with tap water, the NaCLO sterilization 10min of 1%, seed is placed in autoclaving and the diameter 90mm culture dish with two layers of filter paper, add 7ml sterilized water, light culture 2-3d under 25 DEG C of conditions；When the plumelet length on stem reaches 0.5cm, prune plumule with knife blade and partial blade expose growing point after put in the plate being covered with dry filter paper, after room temperature places the moisture that the sterilized filter paper bar of 5min siphons away stem apex spilling, room temperature places 20min again；
B, the infecting and co-culture of receptor: Takara company is synthesizedTCMT3Gene DNA fragment has passed through intermediate carrier pAHC25 at TCMT3 gene two ends plus ubi-1 promoter and no terminator, is then implemented in the plasmid Pcambia0390 containing T-sequence again, is finally transformed in agrobacterium strains EHA105；, design inspection exogenous geneTCMT3Special primer transformant is detected, finally utilize " Micro trauma bud growing point Agrobacterium-mediated Transformation technology " to arabian cron bud growing point import exogenous geneTCMT3；The Agrobacterium of single bacterium colony is taken with aseptic Tip choicest, access containing in antibiotic LB liquid medium, in 28 DEG C, 220rpm when be cultured to bacterial concentration be OD600=0.6,4,000rpm, 5min is centrifugal collects thalline, 3 times that are resuspended in original bacteria liquid volume are infected in liquid, this infects liquid containing 100 μm of ol/LAS, 100mg/LF68,1/10MS salt, 1% glucose, 4% maltose, PH=5.6；Dipping with transgenic brush after infecting liquid alignment growth pricking method brush two to three times, be placed in the sterile petri dish of diameter 90mm, this sterile petri dish, added with sterilizing filter paper 2 layers, sterilized water 0.5ml, is placed under 24-25 DEG C of condition light culture 2-3d；
C, the cultivation of transformation seedlings and management: after co-culturing end, proceed in the culturing pot equipped with Vermiculitum soil by receptor, in 20 DEG C, the illumination box of 16h/d is cultivated intermediate house after 7d, after maturation, individual plant results, only gather in the crops main fringe；
D, transformation generation screening: each for the material of results main fringe is randomly selected 30 kinds respectively and enters in culturing pot, put into greenhouse；When seedling length a to leaf wholeheartedly time, smear blade with 20mg/LPPT, after 7d, pull out non-resistance Seedling, resistance transplantation of seedlings is entered in flowerpot, further exogenous gene is carried out Molecular Detection to determine transgenic seedling.
Adopt and have the beneficial effects that produced by technique scheme:
The hyperaccumulative plant having now been found that only extracts certain metal mostly, and the heavy metal pollution of soil is often the combined pollution of a few heavy metal species, and forefathers study, and the most hyperaccumulative plant Biomass in the nature of discovery are low, poor growth, plant are short and small, limit it and Heavy Metals in Soil Contaminated removed efficiency, be also unfavorable for large-area mechanized operation.The present invention is for the purpose of the overall efficiency improving phytoremediation, research discovery through us, TcMT3 is the major gene of the super accumulation heavy metal of Thalaspi arvense L. platymiscium, pass through transgenic technology, import super accumulation heavy metal characteristic gene (TcMT3) and possess certain heavy metal accumulation ability to itself, Biomass is big, in the Sudan's cursive script of fast growth, cultivate excess of export accumulation type transgenic plant, and on this basis, propose based on phytoremediation, it is aided with animal, the hands sections such as microorganism and Agro-ecology measure, strengthen Lead In Soil, the biological effectiveness of cadmium, promote growth and the absorption of plant, thus improving the effective measures of the comprehensive repair efficiency of over-accumulated plant；This is the innovative technique being different from conventional single repair mode.
The present invention is in the biological synthesis recovery technique of one in conjunction with plant, animal, microorganism and Agro-ecology measure, achieve the biology in situ reparation to soil smoothly, have a significant effect, and environment not being produced secondary pollution, the heavy-metal contaminated soil being following repairs the developing direction in field.The present invention is better than the comprehensive repair technology of single reparation means on repairing effect: as:Jia Chan(Xibei Univ. of Agricultural & Forest Science & Technology；Agro-environment science journal, 6 phases in 2014) and arabian cron can absorb cadmium from cadmium-pyrene combined contamination soil, and the plant clearance of Cadmium in Soil is between 4.23%-17.88%, and the repairing effect of this patent is from slight to serious pollution, and the clearance of cadmium both is greater than 60%.The present invention is better than physical chemistry etc. in environmental friendliness and easily brings the recovery technique of secondary pollution；The physical chemistry restorative procedures such as surface restoration agent and chelating agen such as contrast interpolation hydroxyapatite, potassium chloride, EDTA (Wang Li, 2015；Dong Shanyan, 2002), this will not bring the secondary pollution of soil because of artificial interpolation chemicals, uses based on plant extract, Lumbricus, microorganism etc. itself to the useful biological secreted material of soil to promote, to promote that effect of plant restoration is for auxiliary, for environmentally friendly reparation approach.
It addition, the classification of the present invention and differentiation treatment technology, combining efficient and economic two-fold advantage, it it is the pioneering creation of this area.Test example that the concrete efficacy parameter of the present invention is as detailed below and embodiment.
Accompanying drawing explanation
Fig. 1 is the scene photograph of development test of the present invention.
Fig. 2 is column design sketch and the arabian cron absorption total amount block diagram to Pb, Cd of the slight plumbous cadmium combined pollution geobiont reparation of embodiment 2.
Fig. 3 is column design sketch and the arabian cron absorption total amount block diagram to Pb, Cd of embodiment 3 moderate lead cadmium combined pollution geobiont reparation.
Fig. 4 is column design sketch and the arabian cron absorption total amount block diagram to Pb, Cd of embodiment 4 severe lead cadmium combined pollution geobiont reparation.
Detailed description of the invention
Following example describe the present invention in detail.Various raw material used in the present invention and items of equipment are conventional commercial product, all can pass through market purchase and directly obtain.Wherein, arabian cron adopts grow kind summer, it is preferred to use the transformed variety " holding back indigo plant-the Sudan " of the present invention.Microorganism adopts composite bacteria agent capable composition: be made up of multiple-microorganism floras such as sulfur bacteria, nitrifier, Ammonifying bacteria, nitrogen-fixing bacteria, purchase from high standard biological engineering company limited, product type: Unigrow microbial bacterial agent, uses density: solid fungicide 30 ~ 50kg/hm2Or 2 ~ 3.5kg/ mu, using method: before sowing, keeping soil moisture about 60%, the shallow 3-5cm that turns over mixes.Lumbricus is conventional commercial kind, throws in density: 6.7 × 105Bar/mu larva (about charges seed 1000 for every cubic metre), and the release time, temperature controlled: 18-25 DEG C for after emerging 2-3 days, and soil moisture controls: about 60%.Humic acids (being 10% with the mass ratio of soil): Shijiazhuang humic acid plant produces.
The biological restoration EXPERIMENTAL DESIGN of the plumbous cadmium combined pollution soil of embodiment 1.
Basic condition for examination soil is as shown in the table:
Table 1 is for examination physiochemical properties of soil and content of beary metal
Soil design 4 add level, be respectively as follows: CK, C1(lead 200mg/kg, cadmium 1mg/kg), C2(lead 400mg/kg, cadmium 5mg/kg) C3(lead 800mg/kg, cadmium 10mg/kg).The Pb(NO of respective amount3)2, Cd(NO3)2It is made into the solution of variable concentrations, repeatedly mixs homogeneously with crossing 2mm sieve soil, every basin dress soil 8kg, and apply base fertilizer (each process sowing amount is identical, and N, P close 30kg/ha, K and close 15kg/ha), the plant variety of plantation is:South Africa hybrid Sudan grass seedArabian cron (latin name (Sorghum ×Sudan,SorghumbicolOr × S.sudanense)).
Test sets 96 basins altogether, and four heavy metals add level, repeats for three times, eight process, is respectively as follows:
Process 1(T1): plant (is equivalent to CK)；
Process 2(T2): plant+microorganism；
Process 3(T3): plant+animal；
Process 4(T4): plant+humic acids；
Process 5(T5): plant+microorganism+animal；
Process 6(T6): plant+microorganism+humic acids；
Process 7(T7): plant+animal+humic acids；
Process 8(T8): plant+microorganism+animal+humic acids.
The Sudan. grass Varieties of plantation growth in summer.After planting, grow 10-15 days, after Seedling all sprouts, throw in Lumbricus, vegetation period be 95 days, gather in the crops its overground part, wash out root system simultaneously, under 70 degree drying, weigh the dry weight of overground part and root.Plant samples HNO3-HCLO4Digestion is then with graphite furnace-aas determination cadmium content；Soil total P b, Cd HCl-HNO3-HClO4-HF clears up, and then use graphite furnace-aas determination Lead In Soil, the cadmium content of the use flame-atomic absorption spectrography (AAS) of high-load, low content, compares it and repair difference.
The biological restoration test of embodiment 2. slightly plumbous cadmium combined pollution soil
Result of the test is shown in accompanying drawing 2.As seen from Figure 2, through the repair process of 95 days, the content decline comparatively notable (during Alpha=0.05) of soil slight pollution total Pb, Cd in processing is C1T1, C1T2, C1T3, C1T4, C1T5, these 5 kinds process lower Pb clearance respectively 34.21%, 31.02%, 27.89%, 32.12%, 30.78%, Cd clearance respectively 92.06%, 92.46%, 91.83%, 91.90%, 92.54%, these 5 kinds process after lower reparation Pb, Cd content difference notable (during Alpha=0.05) in soil.In conjunction with Fig. 2 arabian cron, Pb, Cd being absorbed total amount visible, it is 1.14mg that C1T5 processes lower arabian cron to Pb absorption total amount, is significantly higher than other 7 kinds process (during Alpha=0.05), and is 0.15% to the contribution rate of Pb removal amount in soil；Cd is absorbed total amount and is significantly higher than other under C1T1, C1T2, C1T3, C1T4, C1T5 process and processes by arabian cron, and under C1T5 processes, Cd absorbs total amount and reaches 0.21mg, for the maximum in each process, accounts for Cd in soil and removes the 0.22% of total amount.Therefore, C1T1 processes economical and effective, and the best repair that C1T5 process (that is: plant+microorganism+animal) is heavy metal slight pollution soil combines.
The biological restoration test of embodiment 3. moderate lead cadmium combined pollution soil
Result of the test is shown in accompanying drawing 3.As seen from Figure 3, through the repair process of 95 days, in soil Pb repairing effect C2T4, C2T5 process under there is significant difference (during Alpha=0.05), after reparation all the other process between P in soil b content without significant difference；And respectively process Cd content difference notable (during Alpha=0.05) in lower soil.But, after reparation in soil Pb, Cd all under C2T5 processes content minimum, clearance respectively 26.28%, 77.89%.As seen from Figure 3, between different disposal arabian cron to the absorption total amount of Pb, Cd without significant difference (during Alpha=0.05), under wherein C2T5 processes, Pb, Cd absorption total amount is maximum, respectively 0.70mg, 0.49mg, accounts for Pb, Cd in soil and removes the 0.07% of total amount, 0.48%.Therefore, C2T1 processes economical and effective, and the best repair that C2T5 process (that is: plant+microorganism+animal) is heavy metal intermediate pollution soil combines.
The biological restoration test of embodiment 4. severe lead cadmium combined pollution soil
Result of the test is shown in accompanying drawing 4.From fig. 4, it can be seen that under the repair process of 95 days, soil serious pollution each process between in soil Pb content minimum be C3T5 processs, and be substantially less than other and process, repairing effect the best, clearance is 39.18%；What in serious pollution soil, Cd content was minimum is, and C3T1 processes and C1T5 processs, clearance respectively 65.97% and 64.51%, and without significant difference (during Alpha=0.05) between two process.From fig. 4, it can be seen that Pb, Cd are absorbed total amount significant difference (during Alpha=0.05) between different disposal by arabian cron, maximum absorption differs 67% with minimal absorption amount, and wherein C3T1 processes lower arabian cron and Pb absorbtivity is significantly higher than other process；And the absorption total amount C3T1 of Cd is processed and processes apparently higher than other by arabian cron, for 1.01mg, maximum absorption differs 66% with minimal absorption amount.Visible, C3T5 process can reduce Pb, Cd concentration in serious pollution soil preferably, improves the arabian cron assimilation effect to Pb, Cd simultaneously.
Foregoing description is only used as the enforceable technical scheme of the present invention and proposes, not as the single restrictive condition to its technical scheme itself.
Claims (9)
1. a plumbous cadmium combined pollution soil comprehensive organism repairing method, it is characterized in that: for the soil of plumbous cadmium combined pollution, based on over-accumulated plant reparation, it is aided with the microorganism remediation that can improve effect of plant restoration and animal is repaired, strengthen Lead In Soil, the biological effectiveness of cadmium and thus promote growth and the absorption of plant, the overall efficiency of raising contaminated soil remediation.
2. plumbous cadmium combined pollution soil comprehensive organism repairing method according to claim 1, it is characterised in that: the method adopts phytoremediation to add animal and repairs the combined remediation method adding microorganism remediation.
3. plumbous cadmium combined pollution soil comprehensive organism repairing method according to claim 2, it is characterized in that: described phytoremediation adopts arabian cron, described animal is repaired and adopts Lumbricus, and described microorganism remediation adopts the composite bacteria agent capable containing sulfur bacteria, nitrifier, Ammonifying bacteria and nitrogen-fixing bacteria；Described plant, animal, microorganism, combine d bioremediation operating procedure be: arabian cron sow before, keep soil moisture 50%-70% by shallow for the composite bacteria agent capable 3-5cm of turning over mixing be manured into soil；Then, after arabian cron Seedling all sprouts, temperature throws in Lumbricus at 18-25 DEG C and soil moisture when 50%-70%.
4. plumbous cadmium combined pollution soil comprehensive organism repairing method according to claim 1, it is characterised in that: first measure the pollution level of soil, and soil contamination grade delimited as slight pollution, intermediate pollution or serious pollution；If soil belongs to slight pollution, then adopt phytoremediation mode or employing phytoremediation to add animal and repair the combine d bioremediation mode adding microorganism remediation；If soil belongs to intermediate pollution, then adopt phytoremediation mode or employing phytoremediation to add animal and repair the combine d bioremediation mode adding microorganism remediation；If soil belongs to serious pollution, then adopt phytoremediation to add animal and repair the combine d bioremediation mode adding microorganism remediation.
5. plumbous cadmium combined pollution soil comprehensive organism repairing method according to claim 4, it is characterized in that: the demarcation method of described soil contamination grade is as follows: the lead content in soil is measured, if lead content is less than 300mg/kg, then delimit as slight pollution, if lead content is more than 300mg/kg and less than 600mg/kg, then delimiting is intermediate pollution, if lead content is more than 600mg/kg, then delimits as serious pollution.
6. plumbous cadmium combined pollution soil comprehensive organism repairing method according to claim 4, it is characterised in that: the cadmium content in soil is measured, if cadmium content is less than 1.5mg/kg, then delimit as slight pollution；If cadmium content is more than 1.5mg/kg and less than 6mg/kg, then delimit as intermediate pollution；If cadmium content is more than 6mg/kg, then delimit as serious pollution.
7. plumbous cadmium combined pollution soil comprehensive organism repairing method according to claim 5, it is characterized in that: described phytoremediation adopts arabian cron, described animal is repaired and adopts Lumbricus, and described microorganism remediation adopts the composite bacteria agent capable containing sulfur bacteria, nitrifier, Ammonifying bacteria and nitrogen-fixing bacteria；Described plant, animal, microorganism, combine d bioremediation operating procedure be: arabian cron sow before, keep soil moisture 50%-70% by shallow for the composite bacteria agent capable 3-5cm of turning over mixing be manured into soil；Then, after arabian cron Seedling all sprouts, temperature throws in Lumbricus at 18-25 DEG C and soil moisture when 50%-70%.
8. the plumbous cadmium combined pollution soil comprehensive organism repairing method according to any one of claim 1-7, it is characterised in that: described phytoremediation adopts common over-accumulated plant kind or adopts transgenic Sudan. grass Varieties.
9. plumbous cadmium combined pollution soil comprehensive organism repairing method according to claim 8, it is characterised in that: the preparation process of described transgenic Sudan. grass Varieties includes:
A, receptor preparation: choose full neat arabian cron seed, clean with tap water, the NaCLO sterilization 10min of 1%, seed is placed in autoclaving and the diameter 90mm culture dish with two layers of filter paper, add 7ml sterilized water, light culture 2-3d under 25 DEG C of conditions；When the plumelet length on stem reaches 0.5cm, prune plumule with knife blade and partial blade expose growing point after put in the plate being covered with dry filter paper, after room temperature places the moisture that the sterilized filter paper bar of 5min siphons away stem apex spilling, room temperature places 20min again；
B, the infecting and co-culture of receptor: Takara company is synthesizedTCMT3Gene DNA fragment has passed through intermediate carrier pAHC25 at TCMT3 gene two ends plus ubi-1 promoter and no terminator, is then implemented in the plasmid Pcambia0390 containing T-sequence again, is finally transformed in agrobacterium strains EHA105；, design inspection exogenous geneTCMT3Special primer transformant is detected, finally utilize " Micro trauma bud growing point Agrobacterium-mediated Transformation technology " to arabian cron bud growing point import exogenous geneTCMT3；The Agrobacterium of single bacterium colony is taken with aseptic Tip choicest, access containing in antibiotic LB liquid medium, in 28 DEG C, 220rpm when be cultured to bacterial concentration be OD600=0.6,4,000rpm, 5min is centrifugal collects thalline, 3 times that are resuspended in original bacteria liquid volume are infected in liquid, this infects liquid containing 100 μm of ol/LAS, 100mg/LF68,1/10MS salt, 1% glucose, 4% maltose, PH=5.6；Dipping with transgenic brush after infecting liquid alignment growth pricking method brush two to three times, be placed in the sterile petri dish of diameter 90mm, this sterile petri dish, added with sterilizing filter paper 2 layers, sterilized water 0.5ml, is placed under 24-25 DEG C of condition light culture 2-3d；
C, the cultivation of transformation seedlings and management: after co-culturing end, proceed in the culturing pot equipped with Vermiculitum soil by receptor, in 20 DEG C, the illumination box of 16h/d is cultivated intermediate house after 7d, after maturation, individual plant results, only gather in the crops main fringe；
D, transformation generation screening: each for the material of results main fringe is randomly selected 30 kinds respectively and enters in culturing pot, put into greenhouse；When seedling length a to leaf wholeheartedly time, smear blade with 20mg/LPPT, after 7d, pull out non-resistance Seedling, resistance transplantation of seedlings is entered in flowerpot, further exogenous gene is carried out Molecular Detection to determine transgenic seedling.
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| CN110153161A (en) * | 2019-07-09 | 2019-08-23 | 上海建为环保科技有限公司 | The technical method of metallic pollution soil is repaired in a kind of animals and plants collaboration |
| CN110283845A (en) * | 2019-07-29 | 2019-09-27 | 中国科学院地理科学与资源研究所 | A kind of genetically modified plants compound system is applied to the plant restoration method of contaminated soil |
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